Polypeptide synthesis may be either solid-phase peptide synthesis (SPPS) or solution-phase synthesis. Unlike ribosome protein synthesis, solid-phase protein synthesis generally proceeds from the C-terminus to N-terminus. There are several groups of peptides and peptidomimetic compounds characterized by derivatization at the C-terminus of the peptide chain. One of these groups includes peptides having C-terminus secondary amides. Within the category of peptides having C-terminus secondary amides, one of the most important families of pharmaceutical products is GnRH agonist analogues as well as antagonists. This family consists of various peptides such as Leuprolide, Deslorelin, Buserelin, Alarelin, Fertirelin, Histrelin, and other analogues.
Peptides with C-terminus secondary amides have traditionally been prepared by SPPS using Boc/Benzyl chemistry either directly on alkylated amino methyl resin (J. Rivier, et al, IJPPR (1985) 25:414-420) or indirectly on Merrifield resin followed by aminolysis where the C-terminus amino acid of the peptide is attached to the solid support by an ester bond (D. H. Coy et al., Biochemistry (1974) 13:303). Both the direct method and indirect method, however, are plagued with drawbacks. With the direct method, peptide yield is relatively low after acidolysis. The indirect method, in addition to slow reaction times, also can suffer from racemization, is cumbersome, and cannot be used with peptides containing protected glutamic acid and/or aspartic acid because their benzyl esters will also undergo aminolysis. Moreover, Boc-SPPS generally utilizes environmentally unfriendly reagents, such as hydrogen fluoride (HF).
To overcome the problems associated with Boc-SPPS synthesis of peptides, Fmoc-SPPS has gained in popularity because of its use of environmentally safer reagents and comparatively milder reaction conditions. In particular, Fmoc-SPPS peptide synthesis avoids the use of HF. One resin that can be utilized for the Fmoc-SPPS synthesis of peptides with secondary amides is a resin bound amine (A. A. Zompra, et al., Chemistry Today (2006) 24(4):49-51). This resin has also been successfully used for the synthesis of small organic molecules such as secondary amides, ureas, sulfonamides, and guanidines (K. G. Estep, et al., J. Org. Chem. (1998) 63:5300-5301). Amine bound resins, however, have several drawbacks for Fmoc-SPPS synthesis of peptides. For example, the synthesis of secondary amides has been reported to be difficult, while the synthesis of sulfonamides has been reported to be unsuccessful. These supports also require expensive intermediates, multi-step reactions, and/or longer reaction time. Taken together, currently available amine bound supports are generally not practical or economical for the large-scale production of peptides with secondary amides. A need therefore exists for a solid support that is economical to produce, and that can be used for Fmoc-SPPS synthesis of polypeptides in high purity and yield, and in particular, for the synthesis of peptides with C-terminus secondary amides.